Sublethal ROS treatment of young human fibroblast cells in vitro accelerates the rate of telomere shortening and apparent senescence of the cells by as much as 10 fold. These studies will (a) clarify the mechanism(s) by which ROS damage reduces subsequent cellular replicative capacity, and (b) test for a causal relationship between telomeric shortening and the loss of cellular replicative capacity. The kinetics of ROS-induced telomeric shortening will be analyzed using a newer more sensitive technique, quantitative Fluorescence In Situ Hybridization with a peptide Nucleic Acid probe to telomeric repeats. The role of DNA repair in ROS induced telomere shortening will be determined; i.e., is shortening due to direct breakage of DNA, or is it associated with repair of telomeric lesions, anodes it require at least one round of chromosomal DNA replication after ROS insult. Finally, in order to test if the ROS-induced telomeric shortening directly limits cell replication, it is proposed to determine if human fibroblasts over expressing the catalytic subunit telomere (hrT) recover replicative ability following ROS damage. If ROS-induced telomere shortening is casually reducing replicative potential, then this hrT-transfected cell line should restore shortened telomeres and proliferative potential coordinately.